1. Field of the Invention
The present invention relates to a biosensor for quantitating a substrate (specific component) contained in a sample liquid such as whole blood, plasma, serum, urine, fruit juice and the like, with accuracy, speed and ease. More particularly, the invention relates to a glucose sensor for electrochemically measuring a concentration of glucose included in a sample liquid by reacting the glucose with an oxidoreductase which can react with specificity to the glucose.
2. Description of the Related Art
Various methods such as an optical rotation method, a colorimetric method, a reductimetry method and other methods which use different kinds of chromatographies have been developed as methods for the quantitative analysis of a saccharide such as sucrose and glucose. However, none of these methods has sufficiently high accuracy because the specificity thereof against saccharides is not very high. Among these methods, the optical rotation method can be easily conducted but is known to be significantly influenced by the operating temperature.
Recently, various types of biosensors have been developed which can easily quantitate a specific component (substrate) in a sample liquid such as a biological sample and food without diluting and stirring the sample liquid.
For example, Japanese Laid-Open Patent Publication No. 3-202764 discloses a biosensor comprising an electrode system formed on an insulating substrate by screen printing or the like and a reaction layer formed on the electrode system and including a hydrophilic polymer, an oxidoreductase, and an electron acceptor. The concentration of a substrate in a sample liquid is measured by using this biosensor as follows. First, the sample liquid is dropped on the reaction layer of the biosensor, so as to dissolve the reaction layer. This causes an enzyme reaction between the substrate in the sample liquid and the oxidoreductase in the reaction layer, and then, the electron acceptor in the reaction layer is reduced. After the completion of the enzyme reaction, the reduced electron acceptor is electrochemically oxidized, thereby measuring the concentration of the substrate in the sample liquid based on an oxidation current value obtained in this oxidation.
U.S. Pat. No. 5,192,415 discloses a biosensor comprising a hydrogen ion concentration control layer which can optimize the pH of the sample liquid depending upon the type of oxidoreductase contained in a reaction layer without previously adjusting the pH of a sample liquid.
U.S. Pat. No. 5,264,103 discloses a biosensor comprising a main electrode system formed on an electrically insulating substrate and including a working electrode and a counter electrode; a reaction layer including an oxidoreductase; and a sub electrode layer provided with an interval from the main electrode system and including a working electrode and a counter electrode.
These biosensors can be used in a wide range of applications, for example as, a glucose sensor, an alcohol sensor, a cholesterol sensor, or an amino acid sensor by appropriately selecting an oxidoreductase to be contained in the reaction layer.
Among these biosensors, a glucose sensor is generally known to include glucose oxidase as the oxidoreductase. Such a glucose sensor has, however, the following problem. Among isomers of glucose, glucose oxidase reacts to .beta.-glucose alone which exists at a proportion of 63% in the equilibrium state. Therefore, a response current value (i.e., detection sensitivity) obtained by such a glucose sensor is so small that measurement error is large, for example, in quantitating an extremely small amount of glucose.
In addition, when this glucose sensor is used to quantitate polysaccharides, most of the glucose produced by a hydrolase is .alpha.-glucose, and hence, an additional procedure is required, before the quantitation, for isomerizing .alpha.-glucose produced through the hydrolysis into .beta.-glucose by using a mutarotase.
Japanese Patent Application No. 6-291401 (unpublished) relates to a biosensor including both mutarotase and glucose oxidase. In this biosensor, however, the detection sensitivity cannot be sufficiently improved when the total amount of these enzymes is small, and the production cost is increased when the total amount of the enzymes is large. Furthermore, when the concentration of a substrate in a sample liquid is comparatively high, the biosensor including mutarotase and glucose oxidase has lower detection sensitivity as compared with a biosensor excluding mutarotase.